Summary The overall goals of our NIGMS-supported research program have been to delineate the regulatory mechanisms that control signaling by protease-activated receptor-1 (PAR1) and closely-related family members in normal physiology and disease. Our efforts have led to the discovery that ubiquitination of PAR1 promotes p38 mitogen-activated protein kinase (MAPK) signaling from endosomes and not lysosomal degradation. Ubiquitination of the P2Y1 receptor functions similarly. This work further revealed a distinct function for ubiquitination of PAR1 in p38-mediated endothelial barrier disruption. We then discovered a novel lysosomal pathway for PAR1 that is also relevant to other G protein-coupled receptors (GPCRs). Contrary to conventional view, we found that ubiquitination of certain GPCRs and canonical ubiquitin-binding ESCRTs are not required for receptor lysosomal degradation. We then identified ALG- interacting protein (ALIX) and arrestin-related domain containing protein-3 (ARRDC3) as key mediators of GPCR lysosomal sorting. The ALIX-dependent pathway bypasses the requirement for GPCR ubiquitination and is distinct from the canonical endosomal sorting complexes required for transport (ESCRT) lysosomal pathway. The ALIX and ARRDC3 pathway also appears to be dysregulated in cancer. The central premise for the proposed studies is that ubiquitination offers novel and diverse mechanisms for regulation of GPCR biology. A thorough understanding of the mechanism by which key regulators and mediators of ubiquitination regulate GPCR signaling and trafficking is essential for understanding dysregulated mechanisms in disease and for identifying new drug targets. The proposed studies will allow my research group to grasp a thorough understanding of novel concepts that we pioneered - that ubiquitination of GPCRs regulates signaling in normal physiology and is disrupted in disease. Our studies have raised many exciting questions and the most transformative research will be pursued. The p38 MAPK mediates inflammation, however it is not known how GPCR-activated, ubiquitin-driven p38 signaling promotes endothelial barrier permeability, a key feature of inflammation, and will be determined. There is a limited understanding of how GPCR endosomal signaling is regulated and is a challenge. Thus, we will define the mechanisms that regulate GPCR-activated, ubiquitin-driven p38 endosomal signaling. Several GPCRs sort through the ALIX-dependent pathway rather than canonical ESCRT, however, the key steps that segregate GPCRs into distinct lysosomal pathways are not known and will be delineated. ARRCD3 expression is either lost or suppressed in breast cancer, however it is not clear how ARRDC3 impacts PAR1 function or contributes to cancer progression and will be examined. We are well equipped to perform these studies given my laboratory's expertise and experience delineating the function of ubiquitination in regulation of GPCR signaling and trafficking in normal physiology and disease.